Significance Hematopoietic stem cell (HSC) transplantation has proven to be a successful approach for treating disease in patients with congenital lymphohematopoietic disorders. The primary advantage of prenatal transplantation includes fetal immunologic immaturity and the potential for induction of donor-specific tolerance to engraftment of donor cells. Objectives The low percent of engrafted donor cells remains the greatest limitation to using in utero HSC transplantation in which there is no inherent survival advantage for donor HSC. The purpose of the current study was to determine whether multiple transplants with recruited CD34+ human peripheral blood stem cells (PBSC) would be effective in increasing the degree of engraftment in a xenogeneic setting. We also explored whether increased populations of CD3+ cells in the transplant enhanced engraftment and if direct fetal administration of rhSCF+rhG-CSF post-transplant preferentially expands the human cells in the (rhesus) host environment. Results Human PBSC were obtained by pheresis from a human male donor. Collected cells were enriched for the CD34+ population and T-cell depleted. Four fetal rhesus monkeys were transplanted intraperitoneal (IP) via ultrasound guidance on gestational day (GD) 50, 52, 54, and 56. Two of four fetuses were also administered SCF (25 5g/kg)+G-CSF (10 5g/kg) post-transplant IP every 10 days from GD 60-150. Four healthy newborns were delivered at term, and blood and marrow collected at birth, then at 2, 3, 4, and 6 months postnatal age. Analysis for engrafted human cells by PCR for the human Y chromosome was positive in marrow for 4 of 4 newborns, and FACS analysis for CD45 showed engraftment ranged from 0.1-1.7%. Future Directions Studies currently focus on transplanted cell number, repeated injections, percent of donor T-cells, and inclusion of donor stromal cells to further enhance engraftment efficiency. KEY WORDS fetus, tolerance, hematopoietic stem cell, in utero transplantation, chimeras FUNDING NIH Grants AI32299 and HL55175